Chemiluminescent detection of analytes of synthetic or natural origin such as proteins or nucleic acids, as well as other biologic molecules, is a popular assay method. Currently available chemiluminescent systems are sensitive and are not accompanied by the safety drawbacks associated with radioactive detection.
In one such assay, luminescence is achieved by the oxidation of a luminol or isoluminol substrate solution in the presence of an oxidizing agent such as hydrogen peroxide or hydrogen peroxide source, such as perborate, and a peroxidase catalyst, such as horseradish peroxidase (HRP). Another assay is based on the use of a solution containing, as a substrate, a phosphate ester of a 1,2-dioxetane. A chemiluminescent signal (commonly referred to as relative light units, RLU) is elicited by action of phosphatase on the substrate. These assays systems have been primarily practiced as independent methods using separate substrate solutions for the determination of either peroxidase or phosphatase activity, exceptions being those examples noted immediately below.
The utility of enzyme substrate formulations for the detection of multiple enzymes has been recognized previously with respect to the provision of a commodity item serving an either/or purpose. For example, Vector Laboratories sells a chemiluminescent substrate formulation for the determination of alkaline phosphatase (AP) or peroxidase activity called DuoLux based on Lumigen's APS-5 acridan substrate for phosphatase and another substrate for peroxidase. While this formulation may be employed in an either/or situation, the use of the formulation in a combined fashion is not indicated, nor recommended, nor would benefits ascribed to the composition indicate that it would have utility in a combined fashion.
In similar fashion, U.S. Pat. No. 6,068,979 shows a sequential chemiluminescent detection technique for peroxidase and alkaline phosphatase utilizing a dioxetane compound for phosphatase activity and an alkylacridan-9-carboxylate derivative for peroxidase activity. This patent, however, does not show the simultaneous detection of the two enzymes, nor does the patent indicate any synergistic advantages of the formulation in its use in a combined fashion.
It would be desirable to have a single substrate solution that could be used interchangeably in a separate as well as in a combined fashion with either of the two enzymes.